The standard enthalpy and entropy of association for "virtual" substrates and small inhibitors to the primary binding site in forms of alpha-chymotrypsin (Cht) in which catalytically essential residues are chemically modified (i.e., dehydroalanine-195-Cht, N-methylhistidine-57-Cht) shows a significantly more positive enthalpy and entropy of association than to native Cht at pH 7.8. We propose to extend the thermodynamic studies of productive and non-productive binding associations in native and chemically modified Chts to: 1) the binding of peptide substrates, 2) the binding of substrates in which rotational degrees of freedom are constrained, and 3) to inhibitors that are transition-state analogs of the enzymic reaction. The thermodynamics of substrate binding in the presence of salts that effect solvent structure will be studied; and the differences in the abilities of substrates in groups 1 and 2 to order solvent water will be determined from the thermodynamic values found for the transfer of these substrates from aqueous to non-polar solvent. Furthermore, the study of substrate binding to other native and catalytically modified proteolytic enzymes is proposed. The enthalpy and entropy for association to native and modified enzymes with substrates in groups 1-3 will lead to information on the mechanism of substrate activation (substrate distortion, induced fit, and/or substrate freezing) on formation of the non-covalent Michaelis complex between enzyme and substrates, and the role of essential catalytic residues of the active site in productive enzyme-substrate associations. The data will be also informative as to the effect of enzyme and substrate solvation on enzyme-substrate association and the effects of substrate desolvation on the thermodynamics of substrate association to enzyme active sites.